Interactions Study of Co-enzyme-Q0 with Aniline and Pyrrole Using Square Wave Voltammetric Technique

The interactions of Co-enzyme Q0 with Aniline and Pyrrole were studied in aqueous phosphate buffer solution at (pH=7.0) as supporting electrolyte using square wave voltammetry, Co-enzyme Q0 gives a well-defined square wave voltammetric peak at (-0.0415) volt against the reference electrode (Ag/AgCl/3M KCl). The binding constants (K) were calculated at different temperatures. Vant's hoff equation is applied to calculate the thermodynamic parametrs (∆H enthalpy changes, ∆S entropy changes and ∆G free energy changes), and then the type of interaction was estimated. The results indicated that the interaction between Co-enzyme –Q0 and Aniline (∆H and ∆S negative values) was probably due to Vander Waals forces or hydrogen bonding interaction, and the second interaction between Co-enzyme-Q0 with Pyrrole (∆H and ∆S positive values) might be due to the hydrophobic interaction.


INTRODUCTION
The chemical structure of coenzyme Q 0 (CoQ 0 ) is C 10 H 12 O 4 , 2,3-dimethoxy-5-methyl -1,4benzoquinone, (Al-Nuri et al.,2011). Fig. (1) Coenzyme Q (CoQ) also known as Ubiquinone (UQ), is a redox-active lipophilic molecule that is found in all cells and in the membranes of many organelles where it participates in a variety of cellular processes (Turunen et al., 2004). In the inner membrane of mitochondria, (UQ) diffuses freely and is necessary for the function of the electron transport chain (ETC), as it enables the transfer of electrons from mitochondrial complexes I and II to mitochondrial complex III. Thus, (UQ) is necessary for appropriate mitochondrial adenosine triphosphate (ATP) generation. UQ is also believed to be critically involved in mitochondrial reactive oxygen species (ROS) generation, like semi ubiquinone generated during electrons transport which can react with oxygen molecule to form the superoxide anion. In its reduced form, UQ has been suggested to be an effective antioxidant, protecting cellular membranes from lipid peroxidation .UQ has an aromatic head group and a polyisoprenoid side chain varying in length between species. The side chain comprises 10 subunits in humans (UQ10) and 9 in mice (UQ9), where UQ10 is also present as a minor species (Wang et al.,2015).
The electrochemical properties of CoQ10 have attracted attention because the CoQ10 participates in a variety of antioxidant reaction. The overall redox process of CoQ10 can be regarded as consisting of a series of consecutive electron-transfers and chemical steps (Michalkiewicz, 2011). It is noteworthy that the pH values have significant effect on the mechanism of the cathodic reduction of CoQ10 (Schrebler et al.,1990;Li et al., 2016) CoQ10 + H+ e ↔ CoQ10 H• CoQ10 H• + H+ e ↔ CoQ10 H2 Moreover, the present redox process that occurs in two one electron, one-proton stages is irreversible. However, the detailed electrochemical mechanism of CoQ10 has remained elusive due to the different electrodes and complicated medium. It is known to us that the metal electrodes can be used to explore the mechanism of biological redox cycling of bioactive molecules (Slawomir, 2007).

EXPERIMENTAL Apparatus
All experiments were performed using 797-VA Computerize stand (Metrohm AG, CH-9101 Herisav, Switzerland ). Reference electrode (RE) was Ag/AgCl, with 3M KCl and Pt wire was used as an auxiliary electrode (AE) and hanging mercury drop electrode (HMDE) was used as working electrode (WE). pH measurements were performed by using a digital pH meter (HAVANNA ) calibrated with standard buffers. HAAKE G water bath was used for controlling temperature during experiments.

Reagents Chemicals Pure Coenzyme Q 0 Solution (10 -3 M):
A stock solution of co-enzyme-Q 0 was prepared by dissolving 0.0018 gm of pure Coenzyme -Q 0 (supplied by Fluka) in 10 ml absolute ethanol.

Aniline and Pyrrole:
Aniline and Pyrrole monomers were distilled twice under vaccum pressure and stored in the dark bottle before use.

Phosphate buffer solution (P.B.S.):
Phosphate buffer was prepared by mixing certain amounts of 0.2 M of each of Na 2 HPO 4 and NaH 2 PO 4 solutions.

Procedure:
Voltammetric technique measurements were used to study the interaction of co-enzyme-Q 0 with aniline and to calculate the binding constant (K), the sample cell contained (10ml) of phosphate buffer at (pH=7.0) with a final concentration (9.9x10 -7 ) M of co-enzyme -Q 0 . The square wave voltammogram was recorded for co-enzyme-Q 0 under the measured optimum conditions ( Table 1).The appropriate amount of (1.1x10 -4 M) of aniline was added to the cell and the square wave voltammogram was recorded at different temperatures in the range (289-308) 0 K in order to calculate the thermodynamic parameters ∆H, ∆S and ∆G. The same procedure was used to calculate the binding constant (K) and thermodynamic parameters of the co-enzyme-Q 0 with (1.44x10 -4 M) of pyrrole.

Table 1: The measured optimum condition of Co-enzyme-Q 0 using SWV technique
The calibration curve of Co-enzyme-Q 0 was constructed using SWV under the optimum condition+ns in Table (1). The square wave voltammograms were recorded for serial additions of (10 -4 ) M co-enzyme-Q 0 in (10 ml) P.B.S. (pH 7.0), and the peak current at Epv (-0.0415 V) plotted against the co-enzyme-Q 0 concentration is shown in Fig. (3). The calibration gives two straight lines depending on concentration rang ,the first one in the concentration range (9.99x10 -8 -1.38x10 -6 ) M with correlation coefficient (R= 0.9948), and the second in the concentration range (1.96×10 -6 -9.5×10 -6 ) M with correlation coefficient (R= 0.9931), this maybe due to the molecular association at high concentrations .
For voltammetric behavior of co-enzyme-Q 0 in the presence of aniline, the square wave voltammogram of (9.9 × 10 -7 ) M of co-enzyme-Q 0 in phosphate buffer at (pH = 7.0) was recorded at (289 0 K). Successive amounts of aniline were then added and the square wave voltammograms was recorded after each addition., The results are shown in Fig. (4). Whereas: Ip1º = peak current for Co-enzyme-Q 0 in the absence of Aniline. Ip1 = peak current for Co-enzyme-Q 0 in the presence of Aniline. Ip2 = A small peak current appeared after addition of Aniline to the Co-enzyme-Q 0 .

Optimum Conditions Values Default Conditions Values Variables
The peak current Ip1 0 of co-enzyme-Q 0 at Epv (-0.0415 V) decreased gradually with the addition of aniline Ip1 until it reached constant value. Weak peak current Ip2 appeared as a reduction wave at (-0.284V) increased marginally to a certain extent (until reaches constant value) with a gradually added of few amounts aniline. This behavior may be due to the interaction of co-enzyme-Q 0 with aniline. The same procedure was applied to the interaction of co-enzyme-Q 0 with Pyrrole. The results are shown in Fig. (5). Where as: Ipº = peak current for Co-enzyme-Q 0 in the absence of Pyrrole. Ip = peak current for Co-enzyme-Q 0 in the presence of Pyrrole.
The peak current Ip 0 of co-enzyme-Q 0 at Epv (-0.0415 V) was found to be decreased gradually with the additions of pyrrole Ip. This behavior is due to the interaction of co-enzyme-Q 0 with pyrrole until it reaches constant value.
To calculate the thermodynamic parameters by plotting ln K against 1/T using Vant Hoffe equation gives a linear relationship as shown in Figs. (8) and (9).

Fig. 9 : The relation between Ln K and 1/T 0 K for interaction between Co-enzyme-Q 0 and Pyrrole
The change in enthalpy (∆H) was obtained from the slope ∆H= -Slope R, and other thermodynamic parameters (∆G and ∆S) were calculated as follows: From which the thermodynamic quantities of the interaction of Co-enzyme-Q 0 with aniline (and pyrrole) can be calculated as shown in (Table 2 and 3).  According to the estimated K-values in (Table 2), the calculation of the enthalpy change (∆H) shows negative value (exothermic),and the temperature rise causes as expected a decrease in the Kvalues of the interaction of co-enzyme-Q 0 with aniline. The calculated free energy changes (∆ G) of this interaction were negative values indicating the possible spontaneous process at the mentioned experimental conditions. The negative values of entropy change (∆S) indicates that the complex formed after the interaction of co-enzyme-Q 0 with aniline is more ordered. From the (Table 3) it is clear that the K-values of the interaction of co-enzyme-Q 0 with pyrrole was found to increase with increasing temperature and the enthalpy change (∆H) showed positive value (endothermic).The positive value of entropy change (∆S) indicates that geometrical configuration after the interaction of co-enzyme-Q 0 with pyrrole is more in disorder. The calculated free energy changes (∆ G) of this interaction were negative values indicating the possible spontaneous process.

CONCLUSION
From the viewpoint of thermodynamics, ∆H > 0 and ∆S > 0 imply that a hydrophobic interaction is the main force; ∆H < 0 and ∆S < 0 reflecting vander Waals forces or hydrogen bonding ∆ H< 0 and ∆S > 0 suggests electrostatic forces that play a key role (Zhao et al., 2010).
Hence from the results in (Table 2), the interaction of co-enzyme-Q 0 with aniline is vander Waals forces or hydrogen bonding while the interaction of co-enzyme-Q 0 with pyrrole is a hydrophobic interaction according to the results obtained in (Table 3).